Quantitation of fiber growth in transplanted central monoamine neurons

Summary Nerve fiber production by central noradrenaline (NA), dopamine (DA), and 5-hydroxytryptamine (5-HT) neurons was studied using immature brain tissue containing locus coeruleus, substantia nigra, or ventro-caudal medulla oblongata respectively, homologously grafted to the anterior chambers of rat eyes. A method was developed for quantitation of the fiber growth that occurs on the sympathetically denervated host irides as observed in whole mounts using Falck-Hillarp fluorescence histochemistry and by the uptake of ³H-metaraminol into the irides. Survival and growth in oculo of the three different areas were characterized by direct observations through the cornea in vivo for a number of pre- and postnatal stages of development of the donors, and the findings correlated to the degree of monoamine nerve fiber production on the host irides. The growth of fetal locus coeruleus transplants on irides was quantified using both fluorescence microscopical measurements of innervated areas and uptake of ³H-metaraminol. The uptake was well correlated to the histochemical measurements on individual irides, thus validating the fluorescence microscopical measurements of fiber production. The fiber growth of fetal locus coeruleus grafts on irides was followed for 20 weeks. The nerves increased in number and uptake capacity approximately linearly for 6 weeks whereafter the increase rapidly levelled off. On average, the final amount of nerve production by fetal locus grafts did not cover more than 1/3 of the host iris surface, and the average uptake of ³H-metaraminol by these nerves did not exceed 60% of that found in sympathetically intact control irides. The locus grafts produced a similar amount of fluorescent fibers in the host iris independent of the crown-rump length stage of the donor fetus and the final size of the transplants in oculo.The survival and growth of NA, DA and 5-HT neurons grafted from various postnatal donor rats was also followed by fluorescence microscopy. Locus coeruleus grafts produced markedly more fibers than the two other types of grafts when the donor was one week old or less, and DA grafts produced the least fibers of the three. Even from one month old donors some MA neurons survived grafting. Also, using prenatal donars, the locus coeruleus grafts produced many more fibers on the irides than did the DA grafts. It was concluded that the intraocular transplantation technique is very suitable for quantitative studies of nerve fiber production by immature monoamine neurons, and that it should be possible to study many other neuron systems in similar ways with this technique.Supported by the Swedish Medical Research Council (04X-03185), Magnus Bergvalls Stiftelse and Karolinska Institutets Fonder. The skilful technical assistance of Miss Ingrid Strömberg, Miss Maud Eriksson and Miss Gerd Boëtius is gratefully acknowledged. Thanks are due to Swedish Pfizer for the generous supply of Nialamid^®     

Distribution of indolealkylamine nerve terminals in the ventricles of the rat brain

Summary The density, distribution and the pharmacologically produced changes of a formaldehyde-induced yellow supra-ependymal fluorescence in the lateral and third ventricles and in the aqueduct of the rat brain are described. The fluorescence consists of small spots or a thin spotted layer just above the ependymal cells. The highest fluorescence densities occur in the areas near the tela chorioidea of the third ventricle and in the interventricular foramen. A high to moderate density occurs in the lateral ventricles and in the aqueduct. Little or no fluorescence is seen above the hypothalamic areas bordering the third ventricle. The fluorescence rapidly fades upon irradiation with violet-blue light, disappears after treatment of the rats with reserpine or p-chlorophenylalanine, is intensified after nialamide or reserpine + nialamide, and does not change after -methyl-p-tyrosine.Electron microscopically supra-ependymal varicose nerves containing small (500 Å) and large (1000 Å) vesicles in the varicosities are observed in areas with supra-ependymal yellow fluorescence. A fine-structural cytochemical technique reveals the presence of a specific, chromaffine, reserpine-sensitive electron dense core in the small and large vesicles.The conclusion is drawn that a characteristically distributed population of supra-ependymal efferent nerve terminals containing an indolealkylamine, most probably 5-hydroxytryptamine, exists in the cerebral ventricles of the rat brain.The skilful assistance of Mr. R. Wybrecht and Mrs. G. Gschwind is gratefully acknowledged.     

5-HT nerve terminals in the fourth ventricle of the rat brain: Their identification and distribution studied by fluorescence histochemistry and electron microscopy

Summary The occurrence and distribution of supra-ependymal nerve terminals storing serotonin (5-HT) are described for the fourth ventricle of the rat brain. The nerve terminals were identified as monoaminergic 1) fluorescence-histochemically, by the presence of a varicose, formaldehyde-induced fluorescence (FIF) on the free surface of the ependyma, 2) electron microscopically, by the presence of electron dense (chromaffin) cores in small (50 nm) and large (100 nm) vesicles found within the varicose regions of supra-ependymal nerve fibres, and 3) by the absence of both the FIF and chromaffin dense cores after treatment with reserpine. Moreover, the serotonergic nature of these nerve fibres could be concluded from 1) the yellow colour of the FIF, 2) the increased FIF after treatment with nialamide or reserpine+nialamide, 3) the diminished FIF and absence of chromaffin dense cores after treatment with p-CPA, and finally 4) the persistence of the FIF and chromaffin dense cores after treatment with -MPT.A high density of 5-HT nerve terminals occurred throughout the floor of the fourth ventricle and on the floor and roof of the lateral recess. Few 5-HT nerve terminals occurred only on the roof of the fourth ventricle (velum medullare, lamina epithelialis of the tela chorioidea), and the surface of the choroid plexus epithelia was devoid of such nerves. Virtually all nerve terminals in the fourth ventricle appear to be serotonergic.Results presented in part at the Autumn Meeting of the British and Italian Pharmacological Societies, Bristol, 1974 (Lorez et al., 1974). The skilful assistance of Mr. R. Wybrecht, Mr. R. Reese and Mrs. M. Gschwind is gratefully acknowledged.     

Dopamine and noradrenaline neurons projecting to the septal area in the rat

Summary The organisation of the catecholamine innervation of the rat septal area was investigated by means of the glyoxylic acid fluorescence method in combination with dopamine uptake studies, lesions and retrograde tracing of horseradish peroxidase. The following catecholamine systems to the septum could be established:The Locus Coeruleus Noradrenergic System These axons are widespread in the septum forming a moderately dense innervation in the anterior hippocampus, the medial septal nucleus, the nucleus of the diagonal band, and the interstitial nucleus of the stria terminalis, and a sparse innervation in the lateral septal nucleus and the septofimbrial nucleus.The Medulla Oblongata Noradrenergic System This system originates in the Al, A2 or A3 cell groups, the axons forming a very dense innervation in the ventral part of the interstitial nucleus of the stria terminalis, a moderately dense innervation in the nucleus of the diagonal band and lateral septal nucleus, and a sparse innervation in the medial septal nucleus, the septofimbrial nucleus and the dorsal part of the interstitial nucleus of the stria terminalis.The Mesencephalic Dopaminergic System This system originates in the medial part of the A10 cell group, the axons forming two distinct terminal patterns. In the first type, smooth axons form pericellular arrangements around non-fluorescent neurons in the lateral septal nucleus. The second type is formed by fine-varicose axons which form a dense band around the fornix in the medial part of the lateral septal nucleus.The Incerto-Hypothalamic Dopaminergic System These axons most probably originate in cell bodies of the diencephalic A11, A13 and A14 cell groups, and are found in the lateral septal nucleus at the level of the anterior commissure.     

De- and regeneration of the bulbospinal serotonin neurons in the rat following 5,6-or 5,7-Dihydroxytryptamine treatment

Summary In an attempt to determine the conditions which permit central 5-HT neurons to respond to a chemical injury of their axons by sprouting and regeneration, the pattern and time-course of recovery of 5-HT concentrations and regrowth of bulbospinal 5-HT axons were evaluated in rats subjected to intraventricular treatment with either 75 g 5,6- or 150 g 5,7-DHT. While 5,6-DHT treatment is followed by a significant recovery of 5-HT concentrations in the telodiencephalon, brainstem and upper part of the spinal cord within 3 months, there is no significant restoration of the severely depleted 5-HT levels in the telodiencephalon and spinal cord, and only limited recovery in 5-HT content of the brainstem preparation after 5,7-DHT.These differences conform to the observation of widespread and effective regrowth and regeneration of the bulbospinal 5-HT neurons in the 5,6-DHT treated lower brainstem and upper spinal cord but restricted and localized sprouting efforts in the 5,7-DHT treated lower medulla oblongata. This could be explained by a cell body near lesion of the non-terminal indoleamine axons by 5,7-DHT which results in a late retrograde, irreversible degeneration of most of the indoleamine pericarya from group B1 and many of group B3.It is concluded that the preservation of a critical length of the main axon and part of its collaterals is necessary for the neuron's survival, and that the individual pattern of the neuropil architecture of brain centres which are invaded by the axonal sprouts may significantly influence their growth characteristics and thus either favour or impede their chance to reestablish connections with their original effector. Aberrant, localized, intense sprouting of drug-damaged axons may in itself reflect the need of the neuron—deprived of most of its axonal tree—to reestablish its original total axonal length by multiple branching.Supported by grants from the Deutsche Forschungsgemeinschaft. The authors are indebted to Rolf Franck for his technical assistance.Supported by grants from the Swedish Medical Research Council (No. 04 X-3874 and 04 X-56).     

Sympathetic ganglia in culture

Summary The accessory cells of rat, guinea-pig and chick sympathetic ganglia have been studied in tissue culture with phase-contrast microscopy, time-lapse cinematography and fluorescence histochemistry. Small intensely fluorescent (SIF) cells have been described for the first time in culture. Satellite cells, which are closely associated with nerve cell bodies, and interstitial cells, which are dispersed throughout the ganglion, appeared to be identical cell types; they do not correspond to oligodendrocytes of the central nervous system as has been suggested previously. However, a small number of cells closely resembling central oligodendrocytes were present. Astrocyte-like cells have also been seen occasionally, although their identity is not clear. Several other cell types were present and have been identified as Schwann cells, sheath cells, fibroblasts, perineural epithelium, macrophages and endothelial cells.This work was supported by the National Heart Foundation of Australia and the Australian Research Grants Committee. We are particularly grateful to Janet McConnell for her excellent technical assistance.     

Formaldehyde induced catecholamine fluorescence in the mouse inferior laryngeal paraganglion

Summary The presence of high concentrations of catecholamines is shown in the mouse's inferior laryngeal paraganglion by means of fluorescence histochemistry. In mice, the entire organ is composed of 20 to 25 small, intensely fluorescent cells of oval shape (about 15 m in diameter). The paraganglion is well provided with capillaries. The identification of catecholamines in the inferior laryngeal paraganglion, originally described as nonchromaffin (parasympathetic) paraganglion, presents additional evidence that all paraganglia store biogenic amines, are related to the sympathetic nervous system, and belong to the APUD cell series.Supported by the Deutsche Forschungsgemeinschaft, Project No: Bo 525/1     

Light- and electron microscopic localization of vasopressin or a vasopressin-like substance in the neurons of the rat suprachiasmatic nucleus

Summary In the suprachiasmatic nucleus of the rat light microscopic immunostaining for vasopressin reveals a distribution pattern of the immunoreactive material different from that known for the supraoptic nucleus. Among non-stained neurons positive-reacting perikarya display a cap- or tiplike labeling. The area of the suprachiasmatic nucleus is marked by delicate vasopressin-positive fibers. At the ultrastructural level the reaction product, after incubation with anti-vasopressin, is localized in small elementary granules unevenly distributed over the cytoplasm. Groups of axons containing specifically labeled granules contact non-reacting fibers.Supported by the Deutsche Forschungsgemeinschaft (Grant Nr. Kr. 569/2) and Stiftung Volkswagenwerk     

Electronmicroscopic immunocytochemical study on the vasopressin-containing neurons of the thirsting rat

Summary Whereas in thirsting animals the perikarya of the nucleus supraopticus are nearly empty of neurosecretory granules as evidenced by electron microscopic observation, the perikarya are heavily stained by light microscopic immunohistochemical staining. In an attempt to discover the substrate responsible for the positive immunohistochemical staining in thirsting rats, the neurons of the supraoptic nucleus of normal and long-term thirsting animals were compared by electron microscopic immunocytochemistry (indirect PAP-method). In controls all parts of the vasopressin-synthesizing neuron are filled with elementary granules which render a positive and uniform reaction after immunostaining with the indirect PAP-method. The positively reacting fibers in the external zone of the median eminence contain smaller granules than those of the tractus supraoptico-hypophyseus. Within the nucleus suprachiasmaticus, no positive reaction after immunostaining was found. In long-term thirsting animals PAP-complexes as markers of vasopressin are located over the ergastoplasm and over the few small elementary granules. The processes within the nucleus supraopticus and the ballooned axons in the internal zone of the median eminence exhibit free, i.e. non granule-bound, PAP-complexes. Findings in the nucleus suprachiasmaticus and the median eminence of thirsting animals correspond to those in controls. The neurohypophysis is almost completely devoid of PAP-labeled elementary granules.From these results it can be concluded that during thirst vasopressin synthesis is increased in the ergastoplasm and that the hormone is transported partly in a non granule-bound form. Direct contacts between neurosecretory cells and the basal lamina are found more often in thirst-stressed animals and are typical of neurohemal regions. It is discussed whether these neurohemal regions may develop transitionally under stress.Supported by the Deutsche Forschungsgemeinschaft (Grant Nr. Kr 569/1) and Stiftung Volkswagenwerk. This work was presented in part at the 72nd meeting of the Anatomische Gesellschaft, Aachen 1977     

Cerebrospinal fluid-contacting neurons in the paraventricular organ and in the spinal cord of the quail embryo: a fluorescence-histochemical study

Although the cerebrospinal fluid-contacting neurons of the avian paraventricular organ exhibit considerable amounts of catecholamines, they show no tyrosine hydroxylase immunoreactivity. In the quail embryo, the development of these neurons has been studied using the paraformaldeyde-glutaraldeyde method for the fluorescence-histochemical localization of catecholamines. The timing of the appearance of catecholamine fluorescence in cerebrospinal fluid-contacting neurons and that in catecholamine-containing neurons of the brainstem have been compared. The first neurons displaying catecholamine fluorescence are found within the locus coeruleus and the nucleus subcoeruleus ventralis on the 5.5th day of incubation. Catecholaminergic neuronal groups of the medulla and mesencephalon can be identified by embryonic day 7, and fluorescent cerebrospinal fluid-contacting neurons of the hypothalamic paraventricular organ can be first recognized at the 8th day of incubation. If the catecholamine content of cerebrospinal fluid-contacting neurons that lack tyrosine hydroxylase depends upon an uptake mechanism, it may be significant that, in fluorescence-histochemical preparations, these neurons can be identified 1–3 days later than those in which catecholamines are synthesized and from which catecholamines are released at an earlier developmental stage. Moreover, cerebrospinal fluid-contacting neurons that have previously been shown to be tyrosine-hydroxylase immunoreactive, and that lie at the spinal-medullary junction display a different developmental pattern. By fluorescence histochemistry, they can be detected only by embryonic day 10.5. The chemical, developmental and topographical differences suggest that the catecholamine-containing cerebrospinal fluid-contacting elements of the paraventricular organ and those of the spinal cord represent two different subsets of cerebrospinal fluid-contacting neurons whose respective functional roles remain to be investigated.     

Morphological aspects of the hypothalamic-hypophyseal system

Summary Enzyme-histochemical methods were used to study the metabolic activity of specialized ependyma of the ventrolateral walls and floor of the third ventricle in young male and female rats during the critical period of sexual differentiation of the hypothalamus (one week after birth). Histochemical tests were conducted for glutamic dehydrogenase, lactic dehydrogenase, glucose-6-phosphate dehydrogenase, glycerophosphate dehydrogenase and NADH₂-dehydrogenase. Enzyme activity was judged by cytospectrophotometry. All the data were treated statistically.It was found that the specialized ependyma of the ventrolateral wall and floor of the third ventricle (median eminence) in rats differed in their enzyme behaviour in males and females during the critical period of sexual differentiation of the hypothalamus. At the level of the arcuate nucleus ( ²-tanycytes) and the medial part of the median eminence ( ²-tanycytes) the ependyma was characterized by similar indices of metabolic activity in males and females in the decisive terms of the critical period (days 3, 5, and 7). On day 5 metabolic activity of these cells was reduced both in the males and in the females. Prominent sexual differences in the intensity of the enzyme reactions studied were noted in the ependyma of the lateral parts of the median eminence ( ¹-tanycytes) in the critical period. On day 5 metabolic activity of ¹-tanycytes was reduced in males and increased in females. It is suggested that these differences are caused by the receptor nature of ¹-tanycytes and suggest their implication in the mechanisms of sexual differentiation of the hypothalamus.     

Projection of ventrolateral medullary (A1) catecholamine neurons toward nucleus tractus solitarii

Summary The distribution and interconnections of brainstem catecholamine cell groups thought to be important in cardiovascular control were studied using histochemical and ultrastructural techniques in the rabbit. Lesions and microinjections of horseradish peroxidase (HRP) were made in the nucleus tractus solitarii in the dorsomedial medulla, and in the ventrolateral medulla. After lesions of the dorsomedial medulla the fluorescence intensity of the Al-group of catecholamine neurons was increased, and swollen axons could be seen coursing from the ventrolateral medulla toward the lesions on the same side, but not the opposite side. Most of these axons ran in a band about 2 mm in width, centered at the level of the obex. Electron microscopically, specific cells, identified as A1-catecholamine neurons, showed evidence of chromatolysis after the dorsomedial lesions. Following injection of HRP into the nucleus tractus solitarii, A1-catecholamine cells in the ventrolateral medulla on the same side contained the reaction product. Lesions of the ventrolateral medulla did not produce evidence of a reciprocal projection of A2-catecholamine neurons toward the ventrolateral medulla.Thus axons of the A1-group of catecholamine neurons in the ventrolateral medulla project toward the ipsilateral nucleus tractus solitarii in a relatively compact band at the level of the obex. On the other hand, the A2-group of catecholamine neurons in the dorsomedial medulla does not appear to send projections toward the A1-group.These studies were supported by grants from the National Heart Foundation of Australia and The Life Insurance Medical Research Fund of Australia and New Zealand, and Merck Sharp and Dohme (Australia) Pty Limited     

Distribution of intrinsic nerve cell bodies and axons which take up aromatic amines and their precursors in the small intestine of the guinea-pig

Summary The sites of uptake, decarboxylation and retention of 1-dopa and the uptake and retention of dopamine and 6-hydroxytryptamine in the small intestine of the guinea-pig have been localised histochemically with a fluorescence technique for arylethylamines. In segments of ileum from untreated guinea-pigs only noradrenergic axons are fluorescent; these axons were eliminated by surgical denervation (crushing nerves running to the intestine through the mesentery) or by chemical denervation with 6-hydroxydopamine. In denervated segments of ileum, cell bodies and processes of intrinsic neurons become fluorescent after the injection of 1-dopa, dopamine or 6-hydroxytryptamine and the inhibition of monoamine oxidase, as do cells of Brunner's glands and Paneth cells. About 11% of the nerve cell bodies in the submucous plexus and 0.4% of those in the myenteric plexus become fluorescent. Varicose intrinsic axons which take up amines are found amongst the nerve cell bodies of the myenteric and submucous plexuses. They also ramify in the principal connections of the plexuses, in the tertiary strands of the myenteric plexus, in the deep muscular plexus and contribute sparse supplies of axons to arterioles in the submucosa and to the lamina propria of the mucosa. The axons are resistant to the degenerative actions of 6-hydroxydopamine.It is suggested that the intrinsic amine handling axons are more likely to utilise an indolamine related to 5-hydroxytryptamine than they are to utilise a catecholamine as a neurotransmitter.     

Vacuolated neurons in the hypogastric ganglion of the rat

Summary The vacuolated neurons (VN) of the main hypogastric ganglion of the male rat were studied using the formaldehyde-induced fluorescence (FIF) method for the histochemical demonstration of catecholamines. Microspectrofluorimetry was performed to identify the fluorophores and to quantify the FIF. The thiocholine method (Koelle-Gomori) was used to demonstrate acetylcholinesterase activity. The fine structure of the VN was studied using glutaraldehyde/OsO₄ fixation.(1) In the untreated adult male rat VN represent only a small population of the total number of hypogastric neurons (0.8–1.2%). The vacuoles are similar to those of the VN from the corresponding female ganglion. (2) The VN are considered to be adrenergic due to the nature of their fluorophore, indicating a primary catecholamine. (3) The first VN appear in the hypogastric ganglia at the age of 7 weeks. After testosterone administration to young rats, VN are found at the age of 4 weeks. (4) The basic fine structure of the VN is similar to that of other ordinary neurons of the hypogastric ganglia. (5) The content of the vacuoles could not be identified. (6) Indications of degeneration were not observed in the VN. (7) The VN are interpreted as being a functional stage of the short adrenergic neurons, which are under the control of steroid hormones. (8) Fifteen months after castration, no VN could be found in the hypogastric ganglia, while their number was normal in the corresponding control animals.